Method and means for indicating product deterioration



Patented Mar. 2,

METHOD AND MEANS FOR INDICATING PRODUCT DETERIORATION James dA. Clark, Longview, Wash.

No Drawing. Application October 17, 1949, Serial No. 121,889

This invention relates to a method and means for indicating spoilage or deterioration of food, or biological products, or non-biological products that deteriorate in a way similar to biological products under the influence of time and warmth, and more particularly to a method and means for the visual indication of the deterioration of food and other cold-stored products which are subject to spoilage under improper or under prolonged storage.

This application discloses new matter beyond that of my copending application Serial No. 618,391, filed September 24, 1945, now U. S. Pat ent 2,485,566, which covers the indication of deterioration or spoilage of a product by means of visual changes brought about by bacteria in a suitable substrate in a transparent holder, attached to the product.

With the growing practice of the use of freezing as a means of preserving perishable foodstuffs and the use of low temperatures for preserving biological or other preparations that deteriorate at normal temperatures and their distribution in small packages, it is often necessary to know that the product in the package is fresh at the time of purchase or use. Especially with the advent of precooked foodstuffs there is dangerof improper storage or transit conditions resuling in the development of bacterially and/or enzymatically caused decomposition in the product to a point where its consumption would be unpleasant or even result in serious consequences.

In the case of certain products like fish or even meat, freezing and keeping at moderately low temperatures below freezing, does not prevent deterioration of the flavor of the product after a few months, and unless the storage temperature is very low, after the passage of longer time the product becomes unpalatable, then inedible.

Packaged products which are not hermetically sealed are subject to oxidation by air in or permeating through the container. This-may supplement deterioration caused by any bacteria and/or enzymes present. Both oxidative and enzymatic action increase with temperature in accordance with Vant Hofis rule. Temperature has a similar effect on bacterial action except that its effect ceases at 0., whereas some nonbacterially caused oxidative and enzymatic actions proceed below 0 C. at a sufficient rate to be appreciableafter a prolonged period. With the growing practice of placing small quantities f pe i h n Qd t ..-;su .h-asfr shzfish; m a s and vegetables or vaccines and similar biological 11 Claims. (01. 99-192) preparations, in individual or small packages for distribution, the presence of the covering material and other factors make it difficult for the user to decide upon the state of freshness. Freshness may be reduced by any or all of the three actions mentioned, in accordance. with an integral function of time and temperature, the nature of the product and the properties of the package.

One of the objects of this invention is to provide a simple and sure method whereby the recipient of a package of perishable product, such as foods, biological preparations and the like, can be assured that upon receipt, such product has not been subjected to conditions which would have stimulated the activity of enzymes, bacteria or other degradative systems in the product for a long enough period to have resulted in a substantial deterioration of the product.

It is a further object of the invention to provide for a method of indicating spoilage in a package by inserting or attaching to the package an indicator comprising an enzymatic system which exhibits a change in appearance when the package has been subjected to storage conditionslikely to have substantially deteriorated ordegraded its contents.

Another object of the invention is to provide a simple visual device for indicating whether or not during storage, a package of a perishable product has been subjected to conditions stimulating the degradation of the product for a long enough period to be harmful, or to have resulted in a substantial deterioration of the product.

A further object of this invention is to provide an indicating means having an enzymatic system on a substrate and a pH-change responsive means which changes in appearance under the influence of a predetermined enzymatic activity.

Other objects and advantages of the present invention will appear in this disclosure and in the appended claims.

The foregoing objects are attained by including or attaching to the package of the product an indicator which contains an enzyme and a substrate selected so as to result in a permanent visible change When the package has been subjected to conditions resulting in a permanent degree of deterioration of the product.

A system adapted to indicate a substantial degree of biological or similar degradation within the package consists of a transparent container or cell associated with the package in which cell, enzymatic action analogous to the result of accumulated biological or similar action within the package is visually observable. For instance, such visual observation is afforded by an indicator responsive to pH change caused by enzymatically produced acid or alkali inside the cell changing in color, or by another indicator whose color or appearance changes by reduction, oxidation or other effect of the enzyme on a suitable substrate. In another indicator, enzymatic action may be evigleiitby liquefaction of a solid substrate and in another by the'generation of gas from the substrate. Thus, a biological or analogous activity resulting in a change within the pack aged product, is simulated by an enzymatic. proc: ess visible from the outside of the package.

The cumulated activity of a specifically lected enzyme and substrate whichmayresult in an observable change in theappearance. or the system is governed by its tempe1'ature (approximately in accordance with Vant I-lofrjs rule) and the elapsed time at that temperature (approximately with the square root of the time) and by thefollowing additional factors, which together with the choiceofenzyme and; substratefprovide convenient means for adjusting the i ndicat or to provide a visible change after a predetermined temperature-time integral has occurred, that is, after a predeterminedainount of commerce biological or similar activity has occurred withinthe'package: (a) initial concentration of the enzyme (1)) concentration of any ooutatoro inhibitor present such 'as' a co-ensensor a salt '(c) pH of the substrate (di buffering or delaying means in the indicator system, to absorb the efiect's of a certain amount of enzyrnaticactivityivithout affecting the appear ance'ofthfe' system. Thus, by selecting the system"and varying these factors, I provide siniplefand efiective 'n ieth'ods for indicating the occu-rrence of reactions'similar to biological activity, from a few minutes at low temperatures to several years at normal temperatures.

Gnesirnpl 'form of the present'invention may consist of small area of a suitable absorbent medium such as'blotting paper dyedor impreg} nated avith a suitable pHind-icator, then coated oiniipregnatea with a suitable substrate andan enzyme system capable o fpr'odu'cing eitherfal kali or acid to change the color'ot thepI-i indicator. This arrangement may be enclosed 'by a transparent covering and/orplaced beneath a transparent" Wrapper of a packaged product, where it may beseen. The package and contents are then placed instorage, which be cooled-or frozen as may be'required to preserve the contents or the package forthe desired time. Iffor any reason during storage, the temperature of the package is raised sufficiently for a long enough cumulated time to cause an undesirable cumulated biological or similar activity of the contents of the package, then the-enzyme system indicator also will have had favorable conditionsfor activity. described, the substrate and enzyme as selected and adjusted so thatthe pI-I indicator changes color when the predetermined degree of biological or similaiaac tivity has occurred to the contents. of the pack age, such that they'have become. deteriorated, ofieflavor, unpalatable, orinedible before the col or change of the indicator becomes evident,

An enzyme systemsuitable for the indicator system described in the preceding paragraph is a dilute solution of g lycolase, and as the substrate' a few percent glucose solution preferably thickened with'agar, and buffered with a2 percent pI-I 8.0citrat orphosiphate-soda bufie'r.

4 A litmus indicator would change from blue to red when the enzyme had converted sufiicient oi the hexose to lactic acid. Another form of indicator in a closed glass capsule, containing at one end a solution containing a 1 percent solution of asparagine and a 0.01 percent solution of asparaginase (an amidase), this mixed solut on be ng i k ne th. seat a ban i gelatin t m d l o th ssett s arate th mixed solution from the other end of the capsule, the other and containing an agar-thickened, citrate biifiered (to pH 6.0), brightly colored solution of phenol red. .The. enzyme system upon activation, produces ammonia gas which permeates the gelatin. layer and, depending on the concentration of the buffer in the indicator, will turn its, color irom bright yellow to bright red, after exposure to conditions resulting in a predetermined amount of biological or analogous action to the contents of the package.

Reductases, obtained from tissue, may be used as an indicator, for example with meth ylene blue (under anaerobic conditions) i ch, they willreduce to its colorless base, The temperature reaction may be controlled by the typeand concentration of the mattr ss and enzymes andthe of the system. Peroxidases, c onvenien tly'obtained from seed;- ling sprouts, malt, or horseradish, in th e ence of a little peroxide, will oxidise urge n u ber of substances which may serve as inlclicat 'llhus, gum guaic or guaia coric 'acid turns ue. and o-cresol gives a brown e 7 teen color.

An oxidase system; provides {simple indicator. Apple or pear or peac juice containing their oxidases may be applied to a piece crab; sorbent paper and sealedin a tr arent coin tai-ner under aerobic conditions; pieces, of

fresh fruit so sealed would-"also serve;

the ensemble is frozen also then subsequ" thawed, the fruit juice (or rfiut'picfr ally browns due to'what is believed tdbe enzymatically causedoxidation reduotipn 1 tion. The rate or browning maceration by adding a suitable a eot'suoh as a (ii-111156 c1316; ride, sulphite or ascorbic acid; oi a added tokeep thebl-I ofthe system s1 g from the optimum'-point. Lipases are especially suitable forindicating a flavor deterioration of fish and meat after pr'o longed storage in a frozen condition innate; tion on fats i a alogous at rot tempera-or". As. an example or an indicator of this 'tyii, be following solution isenclos'e'd in atraosoiirtn capsule: a 1.0- percent solution or" a sorbitol es ter of a fatty acid (such esters are man oes-"under. the trade name or a'w eno, czoi prcoridolf pancreatin n. s; R, o; n ao phosphate one or so as-to give thesoluti'on a 'inolarity of about onerfifth and enough litmus 'to give a" desirable depth of blue color. By decreasing the perc t y increasing the fliolari y age of pancr'eatin or of the bufier or its p-Hgth cuifiulative conveyor the enzymemay be ex ridedbefore the lit-mus is turned. red. By"increasing theenzyme or o creasing the bufier oi its pH- sufiicient f-atty acidwill be released. to-turrrthe lit" cumulative onz maaeeoaas; In this manner; the indicatormaybe adjusted so it becomes red after a preselected "degree'or" deterioration of'the contents of the package tdwhichthih dicator has been aflixed, has: been reached'l One of the many-ga'siproducing' enzymes and substrates, such as zymase some ire-rose; on; closedin a'transparent; sealedfiexiblecontalrier u's roe" with oss (e. g'; of vinyl chloride-vinilidene chloride copoly mer material, such as made by Dow Chemical Company under the name Saran) may serve well as an indicator in cases where-the extent of the biological action that .hastaken'place, is desired to be approximately known; the extent will correspond approximately to the volume of visible gas produced. A more exact measure could be made if the substrate were titrated or otherwise analyzed before and after storage. With this procedure there is no need to use a special indicator in the container; the gas generated serves as the indicator. The use of a rigid container would permit a certain amount of action to occur without an indication, the initialgas being dissolved in the liquid substrate; this may be advantageous in many applications As a further example of an indicator suitable for the purpose of this invention, a transparent container may be filled with a mixture of a granulated gel of a protein such as gelatin and some proteolytic enzyme such as pepsin, dry starch or dry powdered sugar, and a trace of dry watersoluble dye powder. The appearance of the dry mixture will be white; however after storage favoring biological activity, the gel particles will be liquefied by enzymatic action and the entire contents of the container will be turned the vivid color of the dyestuff. The extent of the activity within the package before the indicator changes may be regulated as already discussed and in addition by the percentage of water used in the gelled particles and the proportion of powdered starch or sugar mixed with these particles.

The temperature coefficients of the enzymatic indicators at various temperatures (the rate of activity at those temperatures) for most purposes will be sufilciently similar to the temperature coefiicients of the biological product to be stored, so that no secondary adjustment to the formulation of the indicator for this purpose will be necessary. However, if this secondary adjustment is needed the neach of the factors controlling the time-temperature integral, the pH concentration of the enzyme, the buffer, salts and other materials and so forth, may be studied experimentally with respect to its individual effect on the temperature coeificient of the indicator as well as its cumulative activity, and the formulation of the indicator varied accordingly to best suit the particular product. In some instances, a small portion of the product itself may be advantageously employed for at least part of the substrate and/or enzyme for the indicator system.

Enzymatic action is, or may be adjusted so that over a range of normal temperatures, it is similar to many chemical and physical reactions with respect to time and temperature. Thus, enzymatic indicators may be applied in the manner disclosed, not only to foods and biological products, but to other preparations such as emul-v sions or suspensions subject to coagulation, or to other products subject, for example, to polymerization after such products or preparations are stored for a period at a range of normal or low temperatures.

In general, there will not be a sharp change in the appearance of the indicator, so that the degree of deterioration of the product can be assessed as none, slight, or considerable, in accordance with the degree of change of color or appearance of the indicator.

In applying the method and device of this invention to frozen products, the indicators may I appreciable be ni adeand kept "frozen till applied, or made" and'verysoon' applied,- and 'attached to each package of the product prior'to' or just after its contents are frozen.- "If storage conditions sub-.

sequently cause deterioration of the product, they also will have caused analogous activity in the enzyme system of the indicator, to produce an irreversibly observable change and thus'give a danger signal to the ultimate recipient of the product.

Enzymatic systems are particularly suited for use as a-spoilage indicator in accordance with this invention, because a large number of themare obtainable in a pure or a standardized form.

Unlike bacteria, which are subject to mutation and to somewhat uncertain activity, enzymes have a fixed potency and may be very accurately measured out, so that a formula for an indicator for a desired purpose may be reproduced with a high degree of certainty and precision."

While the invention has been described in more or less detail, it is not to be limited to such details, as changes may be made in the procedure, form, arrangement and constructionof parts, and equivalents maybe substituted, without departing from the spirit and the scope of the invention as definedin the appended claims, the forms hereinbefore described being merely preferred embodiments thereof.

Having thus fully described my claim: I r g l. The method of indicating that a product has been under time and temperature conditions favoring appreciable deterioration, comprising placing adjacent to the product an enzyme system and a visual indicatorresponsive to enzyme action, which indicator. assumes achange in ap; pearance after the ensemble has been subjected to time and temperature conditions producing deterioration of the packaged product.

2. The method of indicating that a product has been kept under temperature conditions favoring an appreciable deterioration, comprising placing adjacent to the product an enzyme system substantially inactive under proper coldstorage conditions for said product but increasing in activity with rise in temperature, and an indicator responsive to enzymatic action, which indicator assumes a change in appearance after the ensemble has been subjected to conditions permitting an undesirable degree of deteriora- 3. In a package for a product susceptible to deterioration by time and warmth, an indicator for registering the occurrence of conditions favoring appreciable deterioration of the said product, said indicator comprising a container associated with said package, said container containing an enzyme system and a substrate whose appearance changes after a predetermined cumulative amount of enzymatic action.

4. A packaged product subject to deterioration by storage conditions, and associated therewith a container containing an enzyme and substrate system, the activity of said system being substantially proportional to that of the product at storage temperatures, and means associated with the enzyme system and container to give an indication when a predetermined amount of activity has occurred.

5. A packaged product subject to deterioration with time and warmth in combination with an indicating device for indicating deterioration of the product, the device comprising a cell, said invention, If

cell containing an enz-Bie-;system substantially inactive under properconditions .fcrusaid prodnot but incnea g in activity withrise: in temperature, and a ,ltfirclrangennesponsive dye, whereby when time and temperature conditions fax/era "predetermined deterioration of theypiaoduct, said enzyme system will change the color of. thedye.

6-. A. package for a product susceptible to deterioration with time and warmth, said package comprising an outer covering; for said product and. an indicator adjacent said outer covering, said indicator comprising ,a. transparent sealed cellcarrying. an enzyme, system! substantially in,- active under proper storage. conditions for said product but increasing in actiizity with rise in temperature, and a substrate adapted to change appearance upon substantial activity of said n yme System.

'7. A packaged product subject to deterioration with time and warmth with a. transparent cell containing a liquefying. enzyme in a. solidub. strate said enzyme. in said substrate having an activity with respect to. temperature substan, tially the same as that oftheproduct.

3.v A packaged product snbjectto deterioration withtime. aindv warmth, with atievicev containing a gasproducing enzymev and-substrate having an activity with respectv to temperature, substantially the same as thatof the product, and means incorporated in the device for indicating the quantity of gas saproduced;

.9, Apackagcd product subject. .to deterioration with. time and warmth, with a cell-containing 2. DH changing enzyme and substrate having an activity with. respect. .to temperature substantially the. same as thattof the product, and a pH- changev-responsive dy within the cell which will change appearance; after predetermined time and temperatur conditions.

19;. A packaged. product sttbiect to deterioration with; time-and warmth in con'mination with an indicating device for indicating deterioration of the product, the device comprising a cell. containing a solid substrate and an enzyme. capable Qt 'liqnefying. said substrate, said enzyme being. substantially inactive during :pmp r st r e conditions. but active when conditions: favorable to deterioration of said packaged product. occur, whereby sufiicientactivity of said enzyme insaid substrate will indicate the existence of improper storage conditions by its liquefication.

1L. A. packaged product subiect to deteriora tion with time and Warmth combination with an indicating device for indicating deterioration oi -the product, the device comprising a-cellacontaming. a solid substrate, a soluble dry dye and an enzyme capable of. liquefyin saidsubstrate; said enzyme being substantially inactive during. proper storage conditions but increased in: acty en conditi ns favorable: to deterioration of said: packag d ,pteduct occur, whereby upon sufiicient activity of said enzyme, said substratewilL be iiguefied. and said dye will: b dissolved in the liquefied substrate and thereby indicatetheoccurrence-of improperstoragc.

JAMES DA.

Refezences Cited in the file of this patent UNITED STATES PATENTS Number- Name Date 2,359,052 Scha'rer Sept. 26, I952; 2,460,215 hase- Jan. 25, 1949 2,553,369- Hoffma-n May 15 I951 

1. THE METHOD OF INDICATING THAT A PRODUCT HAS BEEN UNDER TIME AND TEMPERATURE CONDITIONS FAVORING APPRECIABLE DETERIORATION, COMPRISING PLACING ADJACENT TO THE PRODUCT AN ENZYME SYSTEM AND A VISUAL INDICATOR RESPONSIVE TO ENZYME ACTION, WHICH INDICATOR ASSUMES A CHANGE IN APPEARANCE AFTER THE ENSEMBLE HAS BEEN SUBJECTED TO TIME AND TEMPERATURE CONDITIONS PRODUCING APPRECIABLE DETERIORATION OF THE PACKAGED PRODUCT. 